In the field of industrial production of flavoring agents there is a continuous demand for efficient and cost-effective means for synthesizing said flavoring agents. Methylized cinnamic acids and cinnamic acid esters, methylized phenethylamine, and the coupling products thereof, particularly cinnamic acid amides, are examples of such flavoring agents. One exemplary cinnamic acid amide is rubemamine. Rubemamine was identified as a natural material in the plant Zanthoxylum rubescens. Currently, however, no biotechnical method for producing said special substance and the direct precursors thereof has been described.
It is known from the prior art that caffeic acid is converted by means of various caffeoyl-CoA-O-methyltransferases (CcAOMT) or cathechol-O-methyltransferases (COMT) and transgenic microorganisms forming a CcAOMT or COMT (Fellenberg, C. et al. Tapetum-specific location of a cation-dependent O-methyltransferase in Arabidopsis thaliana. Plant J. 56, 132-45 (2008); Ibdah, M., Zhang, X.-H., Schmidt, J. & Vogt, T. A novel Mg(2+)-dependent O-methyltransferase in the phenylpropanoid metabolism of Mesembryanthemum crystallinum. J. Biol. Chem. 278, 43961-72 (2003)). The 3′ position is always thereby modified and exclusively ferulic acid is formed. The methylization of the 4′ position has previously been demonstrated only for monolignoles and phenylpropanoids using a variant of (iso)eugenolmethyltransferase from Clarkia breweri (U.S. Pat. No. 8,889,392 B2, Zhang, K. et al. An engineered monolignol 4-O-methyltransferase depresses lignin biosynthesis and confers novel metabolic capability in Arabidopsis. Plant Cell 24, 3135-52 (2012)) and by means of further O-methyltransferases on phenylpropenes such as chavicol or eugenol (Gang, D. R. et al. Characterization of Phenylpropene O-Methyltransferases from Sweet Basil: Facile Change of Substrate Specificity and Convergent Evolution within a Plant O-Methyltransferase Family. Plant Cell 14, 505-519 (2002). A method for enzymatically permethylizing phenethylamines such as dopamine, 3-methoxytyramine, or 3-hydroxy-4-methoxy-phenethylamine is not known. Converting L-DOPA to dopamine by means of DOPA-decarboxylases and by means of transgenic microorganisms producing said enzymes has been described (Facchini et al. Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications. Phytochemistry 54, 121-38 (2000)). Modifying the resulting dopamine by means of 3′-O-methyltransferases (3-OMTs) is also known, wherein 3-methoxytyramine is formed as a product (Lotta, T. et al. Kinetics of Human Soluble and Membrane-Bound Catechol O-Methyltransferase: A Revised Mechanism and Description of the Thermolabile Variant of the Enzyme. Biochemistry 34, 4202-4210 (1995)). It has further been demonstrated that 4-methoxytyramine is also produced from the deltaproteobacterium Myxococcus xanthus from dopamine by the activity of the enzyme SafC (Nelson, J. T., Lee, J., Sims, J. W. & Schmidt, E. W. Characterization of SafC, a catechol 4-O-methyltransferase involved in saframycin biosynthesis. Appl. Environ. Microbiol. 73, 3575-80 (2007)). Permethylization starting with dopamine, 3-methoxytyramine, and/or 3-hydroxy-4-methoxytyramine and forming 3,4-dimethyoxyphenethylamine, however, has not been demonstrated, neither by enzymatic conversion nor by biotransformation using transgenic microorganisms or fungi.
It was further known that S-adenosylmethionine synthases (SAMSs) can be used for producing S-adenosylmethionine (SAM). The combined expression of O-methyltransferases using S-adenosylmethionine-synthases for improved provision of the cofactor S-adenosylmethionine has previously be demonstrated only for forming flavonoids (Sung, S. H. Optimization of Rhamnetin Production in Escherichia coli. J. Microbiol. Biotechnol. 21, 854-857 (2011)), but not for forming methylized cinnamic acids and phenethylamines. The forming of coenzyme A esters of cinnamic acid by the activity of 4-coumarate:CoA ligases is also known (Lindermayr, C. et al. Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family. Eur. J. Biochem. 1315, 1304-1315 (2002)). Ligation of the formed CoA esters using the amines by means of tyramine-N-hydroxycinnamoyltransferases, however, has been previously demonstrated only for non-permethylized substrates such as ferulic acid and dopamine (Yu, M. & Facchini, P. J. Purification, characterization, and immunolocalization of hydroxycinnamoyl-CoA:Tyramine N-(hydroxycinnamoyl)transferase from opium poppy. Planta 209, 33-44 (1999)). No synthesis method starting with cinnamic acids and L-DOPA is known. Further, the methods known under the prior art do not include any biotechnical method for producing cinnamic acid amides in the sense of the present invention, particularly no method suitable for industrial production of cinnamic acid amides, as the yields are estimated to be very small (e.g., 215 mg/L in Kang, K. & Back, K. Production of phenylpropanoid amides in recombinant Escherichia coli. Metab. Eng. 11, 64-68 (2009)).
Therefore a need for establishing biotechnical methods has arisen, that is, both fermentative and enzymatic methods comprising the use of a recombinant organism at least in one step and being suitable for industrial application, in order to thereby reproducibly provide methylized cinnamic acids and phenethylamines and derivatives and coupling products thereof at a sufficient scale.